As shown in the following Formula [I], hesperidin is a compound where rutinose (L-rhamnosyl-(.alpha. 1.fwdarw.6)-glucose) binds to the hydroxyl group at the C-7 in hesperetin (3',5,7-trihydroxy-4'-metoxyflavanone) via the .beta.-linkage. ##STR1##
Hesperidin is present in immature fruit skins of citruses and used in pharmaceuticals, cosmetics, etc., as a vitamin P that has physiological functions such as capillary protestant, hemorrhagic prevention, blood-pressure control, etc. Hesperidin dissolves in aqueous alkaline solutions but not substantially in water and acids. Because only about one gram hesperidin dissolves in 50 1 water (about 0.002 w/v %) at ambient temperature, it easily causes cloudiness in the liquid parts of canned foods with only a slight amount thereof to deteriorate the products' value.
There have been proposed methods to prevent such cloudiness in liquids induced by hesperidin. For example, Japanese Laid-Open Publication No. 7,593/91 discloses a process for producing an enzyme-treated hesperidin with improved water-solubility, which comprises the steps of contacting hesperidin with a saccharide-transferring enzyme (an enzyme having .alpha.-glucosyl-transferring activity) in the presence of partial starch hydrolyzates (.alpha.-glucosyl saccharides) to form .alpha.-glucosyl hesperidin as shown by the following Formula [II]: ##STR2##
As shown in the above Formula [II], the .alpha.-glucosyl hesperidin is a compound where glucose(s) (Gn, n=1-20) successively bind(s) via the .alpha.-1,4 linkage to the C-4 of the glucose in hesperidin in Formula [I], or a mixture of such .alpha.-glucosyl hesperidins having different numbers of glucoses.
In the enzymatic reaction system, 40-80% hesperidin contained in a material solution is converted into .alpha.-glucosyl hesperidin by the enzyme treatment, while 20-60% hesperidin still remains intact free of reaction. The coexistence of .alpha.-glucosyl hesperidin increases the solubility of intact hesperidin in aqueous solutions, but a higher proportion of the intact hesperidin to the .alpha.-glucosyl hesperidin results in an insolubilization and crystallization of intact hesperidin within a relatively short period of time.
Although it can be proposed a method for adding gelatinizers such as carboxymethyl cellulose (CMC) to aqueous solutions containing hesperidin to increase the viscosity of the solutions as a means to prevent the crystallization of intact hesperidin, it could not be a general method because such an addition of the gelatinizers will not be acceptable in view of product image and should not be used in export products.
Also there exists a method for delaying the crystallization of intact hesperidin by crystallizing intact hesperidin and filtering the mixture to separate and remove the crystallized hesperidin in order to lower the proportion of the intact hesperidin to .alpha.-glucosyl hesperidin. Even with the method, intact hesperidin does crystallize after a relatively long period of time as a demerit, and therefore it could not be a substantial solution.
There still exists a method which comprises a step of collecting only a fraction of .alpha.-glucosyl hesperidin from aqueous solutions containing .alpha.-glucosyl hesperidin and intact hesperidin by a technique such as chromatographic fractionation to obtain a desired product for use. The method, however, results in a cost increase and is far from a beneficial method.
The present inventors continued studying to solve the problems in the prior art and found that an enzyme-treated hesperidin, which has extremely-increased water-solubility and does not substantially cause cloudiness even after a relatively long period of time, is obtainable by contacting an enzyme having .alpha.-L-rhamnosidase activity with solutions containing .alpha.-glucosyl hesperidin and intact hesperidin to hydrolyze intact hesperidin to release rhamnose and to be hydrolyzed into .beta.-monoglucosyl hesperetin in the following chemical formula [III], while .alpha.-glucosyl hesperidin is remained substantially unchanged: ##STR3##
Takashi KOMETANI, Yoshinobu TERADA et al. disclosed in Nippon Shokuhin Kagaku Kogaku Kaishi (Japan Food Science Technology), Vol. 42, No. 5, pp. 376-382 (May of 1995), titled "Formation of Sugar Derivatives of Hesperidin and Stabilization of Natural Pigments by Cyclodextrin Glucanotransferase", that they found that: After reacting hesperidin as a saccharide acceptor, .beta.-cyclodextrin (.beta.-CD) as a saccharide donor, and CGTase as a saccharide-transferring enzyme for obtaining sugar derivatives of hesperidin designated as Hsp-Gn, the action of (.alpha.-rhamnosidase on the sugar derivatives during the purification step thereof results in a selective hydrolysis of intact hesperidin, and this facilitates the effective separation of the sugar derivatives in a purified form from the intact hesperidin on column chromatography using a column packed with "SEPHADEX LH-20"; and that they succeeded to inhibit the color fading of natural pigments induced by ultraviolet by adding the purified sugar derivatives.
However, the method of the above literature is to prepare .alpha.-monoglucosyl hesperidin in the formula [IV] by separating concomitant hesperetin using "AMBERLITE XAD-16" and separating a-monoglucosyl hesperidin and .alpha.-diglucosyl hesperidin from a mixture of the resulting .beta.-monoglucosyl hesperetin, .alpha.-monoglucosyl hesperidin, and .alpha.-diglucosyl hesperidin by using an expensive column chromatography. The method is substantially impossible to selectively separate a high .alpha.-monoglucosyl hesperidin content product in a satisfactorily-high yield. Thus, the preparation of such a product in a simpler manner and at a lower cost will give a much more expectation of being widely used in pharmaceuticals, cosmetics, food additives, etc. ##STR4##
The above-identified Japanese Laid-Open Publication No. 7,593/91 discloses a method which comprises the steps of contacting glucoamylase (EC 3.2.1.3) with a mixture solution of .alpha.-glucosyl hesperidin and hesperidin to hydrolyze the .alpha.-glucosyl hesperidin to .alpha.-monoglucosyl hesperidin, feeding the mixture to a column packed with "DIAION HP-20", washing the column with water, and collecting a fraction of .alpha.-monoglucosyl hesperidin while increasing step-wisely the concentration of ethanol in the aqueous system. Even the above-mentioned method could substantially be impossible to supply a high .alpha.-monoglucosyl hesperidin content product at a satisfactorily lower cost.
After further study the present inventors found that .alpha.-monoglucosyl hesperidin is effectively separated and collected from the mixture obtained in the below by using a method which comprises the steps of either contacting glucoamylase and .alpha.-L-rhamnosidase (EC 3.2.1.40) or contacting glucoamylase, .alpha.-L-rhamnosidase, and .beta.-D-glucosidase (EC 3.2.1.21) with a solution containing .alpha.-glucosyl hesperidin and hesperidin; crystallizing the formed .alpha.-monoglucosyl hesperidin in the resulting mixture; separating the crystals; and collecting the separated crystals.
The present invention produces a high .alpha.-monoglucosyl hesperidin content product in an extremely high yield with only applying a solid-separation method to a starting mixture to separate crystallized .alpha.-monoglucosyl hesperidin.
As a method for preventing the cloudiness of syrups in canned mandarin oranges, a method for improving the water solubility of hesperidin, which comprises contacting an enzyme having .alpha.-L-rhamnosidase activity with hesperidin to convert it into .beta.-monoglucosyl hesperetin, is employed on an industrial scale.
However, there has been no indication of a method like the present invention which comprises the steps of either contacting glucoamylase and .alpha.-L-rhamnosidase or contacting an enzyme preparation having glucoamylase, .alpha.-L-rhamnosidase, and .beta.-D-glucosidase activities with solutions containing .alpha.-glucosyl hesperidin and intact hesperidin to hydrolyze the .alpha.-glucosyl hesperidin to form .alpha.-monoglucosyl hesperidin, where leaving the rhamnose residue in the .alpha.-glucosyl hesperidin unchanged and leaving only one glucose residue among the glucose residues (Gn) that are linked to the C-4 of the glucose residue in the .alpha.-glucosyl hesperidin, and to hydrolyze the rhamnose residue in hesperidin into .beta.-monoglucosyl hesperetin, i.e., 7-O-.beta.-monoglucosyl hesperetin, and optionally further to hydrolyze the .beta.-glucose residue in the .beta.-monoglucosyl hesperetin into hesperetin; effectively crystallizing and separating only the .alpha.-monoglucosyl hesperidin; and collecting the resulting .alpha.-monoglucosyl hesperidin.
Japanese Laid-Open Publication No. 80,177/96 discloses a method for preventing the crystallization of hesperidin by the addition of solubilized hesperidin to aqueous solutions containing hesperidin, where the solubilized hesperidin is a compound that is composed of one to ten and several glucose residues bound to the C-4 of the glucose residue in hesperidin via the .alpha.-1,4 linkage and that is producible by contacting saccharide-transferring enzymes with hesperidin in the presence of cyclodextrins, etc., where the saccharide-transferring enzymes are, for example, CGTase or 1,4-.alpha.-D-glucan; 1,4-.alpha.-D-(1,4-glucano)-transferase (EC 2.4.1.19), and more concretely a saccharide-transferring enzyme prepared from a culture of Bacillus A2-5a strain.
The above publication, however, simply discloses a technical art to prevent the crystallization of hesperidin in products containing the hesperidin by admixing solubilized hesperidin with intact hesperidin. Products, obtained by the method and disclosed in the publication, are mixtures of .alpha.-glucosyl hesperidin and hesperidin, meaning that they still contain intact hesperidin, and as a demerit they easily and gradually cause cloudiness in the liquid parts of canned products. To solve this problem, .alpha.-monoglucosyl hesperidin and .alpha.-diglucosyl hesperidin fractions are collected and used in an Example of the above-identified publication. However, economical collection of the two fractions involves technical difficulties.
The present invention solves the above problems in the prior art and is to provides a method for collecting a high .alpha.-monoglucosyl hesperidin content product by treating solutions containing .alpha.-glucosyl hesperidin and hesperidin with a relatively simpler treatment.